Nucleic acid aptamers are short (˜100 nt) structured oligonucleotides that have been selected from large sequence-diverse libraries and shown to display high affinity and specificity for a wide range of targets ranging from simple metal ions (Ref. 1) to complex surface proteins on living cells (Ref. 2). This combination of properties has led to growing interest in applications of aptamers in fields including therapeutics, chemical analysis, biotechnology, chemical separations and environmental diagnostics (Ref. 3). Aptamers are identified from large libraries of random nucleic acid sequences via an iterative in vitro process called SELEX (Systematic Evolution of Ligands by EXponential enrichment) (Refs. 4-6). A typical SELEX round includes the following three steps: (i) Binding: incubation of the library with the target; (ii) Partitioning: separation of target-bound library sequences from unbound ones; (iii) Amplification: generation of a new pool of nucleic acids by making multiple copies of the sequences that bound to the target. These steps are then repeated in an iterative fashion to obtain an enriched pool and the target binding aptamers are identified via cloning and sequencing processes.
Different selection strategies have been developed to separate or partition the free and target-bound sequences, a critical step to ensure the successful identification of only the strongest binding aptamers. Affinity chromatography is one such partitioning strategy that uses specific binding onto resin-immobilized targets to purify macromolecular solutes from dilute solutions (Ref. 7). It is a well-documented aptamer selection technique given that it can achieve a level of purification greater than 95% in a single step and that numerous types of resin media are available to bind a wide variety of targets. However, there is limited understanding of the relationship governing the process parameters (target loading, resin volume, etc.) and the selection quality, and thus, many selection rounds (typically 12 to 15) are required to identify aptamers with the desired specificity and affinity for the target. This approach is particularly challenging when working with RNA libraries because it takes approximately two days just to complete the amplification step. Some work has been done to parallelize the use of libraries and the selection process to multiple targets in order to save time and reagents (Refs. 8 and 9).
Affinity chromatography-based selections are typically done in two different modes of operation. In the batch-mode, a small amount of target-immobilized resin (˜20 to 200 μL) is incubated with the nucleic acid library or alternatively target-free resin is incubated with a mixed suspension of target and nucleic acid library (Refs. 10-12). Any unbound sequences in the supernatant are removed and the target-bound sequences remaining on the resin are then exposed to other solutions for the subsequent processing steps. For this approach, the entire selection process is quite laborious, because each step must be done manually and repeated several times. This is especially true when multiple targets are considered for selection. The second mode of operation, flow-mode, uses small columns (˜0.5 to 3 mL) packed with resin (Refs. 1, 13, and 14). The primary advantage of this approach is that the resin is physically confined within the column, allowing all of the selection steps to be automated using pumps and/or centrifuges and thus completed more efficiently than the batch strategy. This approach was used in one of the landmark papers on aptamers—Ellington and Szostak (Ref. 5) used a 3.5 mL column filled with dye-immobilized resin. However, there are limitations to this approach. The standard columns that have been used previously are not practical for the simultaneous selection of aptamers for multiple targets. These columns require more resin than the batch-mode, as well as more of the immobilized target (e.g., protein), which can be both limiting and expensive. Thus, with the current affinity-chromatography based strategies, there is a noticeable lack of means to rapidly screen for aptamers to multiple targets in a high-throughput and efficient manner.
The present invention is directed to overcoming these and other deficiencies in the art.